The Epidermal Growth Factor (EGF), which is a 53 amino acid polypeptide with a molecular weight of 6045 D, was isolated and purified for the first time from murine submaxillary gland (Cohen S; J Biol Chem (1962) 237, 1555). Later, a similar molecule was obtained from human urine (Cohen S and Carpenter G; (1975) PNAS USA 72, 1317). The action of this polypeptide is mainly performed via its membrane receptor, a 170 KD molecular weight glycoprotein. Its intracellular domain is associated with a tyrosine kinase activity with structural homology to the oncogene v-erb-B showing relation to the malignant transformation process (Heldin C H et al; (1984) Cell 37, 9-20).
High levels of EGF-R have been detected in malignant tumors originating in the epithelium such as breast, bladder, ovarian, vulva, colonic, lung, brain and esophagus cancers. The role played by EGF and its receptor in regulating tumor growth is unknown, but it has been suggested that the EGF-R expression in tumor cells provides a mechanism for autocrine growth stimulation which leads to uncontrolled proliferation (Schlessinger J, Schreiber A B, Levi A, Liberman T and Yarden Y; (1983) Crit Rev Biochem 14(2), 93-111).
It has been reported that EGF produces overgrowth of breast cancer cell lines (Osborne C K et al; (1980) Cancer Research 40, 2361), besides that it modulates the differentiation under some cellular systems (Tonelli C J; Nature (1980) 285, 250-252). These effects on cellular differentiation and proliferation are related to the high expression of EGF-R (Buss J E et al; (1982) PNAS 79, 2574).
The presence of EGF-R in tumor cells has proven to be an indication of a poor prognosis in human breast cancer. Approximately 40% of the breast tumors show specific binding sites of high affinity for the EGF (Perez R, Pascual M R, Macias A, Lage A; (1984) Breast Cancer Research and Treat 4, 189-193). There is also an inverse correlation with the presence of estrogen receptor, indicating EGF-R as an indifferentiation marker or an indicator of the potential capacity of proliferation of the malignant cells.
Other groups have reported that the expression of EGF-R is higher in regional ganglionar metastasis than in primary carcinomas of breast (Sainsbury J R et al; (1985) Lancet 1, 8425, 364-366) and that the expression of the receptor is different in the different histologic subtypes of human breast carcinoma cells, their presence constituting a signal of bad prognosis (Macias A et al; Anti Cancer Research 6: 849-852).
The evidences obtained in different studies have prompted to consider the EGF/EGF-R system as a possible target for therapeutic actions.
We obtained a murine monoclonal antibody (R3), raised against the human placenta as described in European Patent application No. 93202428.4, and found to bind to the external domain of the human EGF-R. It was found to inhibit the binding of EGF at both low and high affinity EGF-R sites.
Passive immunotherapy using monoclonal antibodies against the EGF-R have been the object of multiple investigations, which have demonstrated that the specific recognition of the receptor by the antibody inhibits the EGF binding, with an inhibitory effect on the mitogenic stimulation of malignant cells (Sato J D et al; (1987) Methods in Enzimology 148, 63-81); but there is evidence that the murine origin of these antibodies produces a human anti-mouse antibody response.
The development of the hybridoma antibody technique by Kohler and Milstein revolutionized the discipline of immunochemistry and provided a new family of reagents with potential applications in clinical diagnosis and immunotherapy (Kohler G, Milstein C; (1975) Nature 256, 495-497). While it has become routine to produce mouse monoclonal antibodies (mAbs) for use in basic research and clinical diagnosis, it has been difficult to use these for in vivo immunotherapy because they have reduced half-life in humans, poor recognition of mouse antibodies effector domains by the human immune system and the foreign immunoglobulin can elicit an antiglobulin response (HAMA response) that may interfere with therapy.
The ability to genetically manipulate antibody genes and then to express these altered genes by transfection techniques enables us to produce mAbs having more desirable properties than the existing hybridoma antibodies. Thus genetic engineering can be used to enhance desired effector functions in antibody molecules and to decrease or eliminate undesired effector functions.
The use of recombinant DNA technology to clone antibody genes has provided an alternative whereby a murine mAb can be converted to a predominantly human form with the same antigen binding properties. S L Morrison in 1984 created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable regions genes of mouse antibody producing myeloma cell lines and joining them to human immunoglobulin constant region (Morrison S L et al; (1984) PNAS USA 81, 6851-6855).
Other authors have attempted to build rodent antigen binding sites directly into human antibodies by transplanting only the antigen binding site, rather than the entire variable domain, from a murine antibody (Jones P T et al; (1986) Nature 321, 522-524; Verhoeven M et al; (1988) Science 239, 1534-1536). Some applications of this method have been developed (Rietchmann L et al; (1988) Nature 332, 323-327; Quee C et al; (1989) PNAS USA 86, 10029-10033), other authors have worked with reshaped antibodies, which included some murine residues in human FRs in order to recover the affinity for the original antigen (Tempest PR; (1991) Biotechnology 9, 266-272).
Orlandi R et al (Proc Natl Acad Sci USA 86, 3833-3837, 1989) disclose the constant regions of the human gamma-1 heavy chain and the human kappa light chain, and suitable cloning vectors therefor.